The immune systems of patients with cancer spontaneously produce antibodies to cancer antigens released by their tumors due to alterations in protein expression, mutation, degradation, or localization. These autoantibodies are potential biomarkers for early cancer diagnosis. Antibodies to a number of tumor-associated antigens have been identified in patient sera, some as early as several years before the clinical appearance of cancer. Although the specificity for these responses is high, typically only 5-20% of patients demonstrate a response to any given antigen, which has limited the usefulness of single antigen responses as biomarkers. By combining the responses to several antigens, tests with markedly improved sensitivity and specificity have been demonstrated for several cancers. The recent development of protein microarrays offers an ideal tool for screening for immune responses to tumor antigens. They have the advantage that hundreds to thousands of different proteins can be printed in the space of a standard microscope slide and only require a few microliters of serum for screening assays. By examining many proteins simultaneously, the concordance of antigen responses can be examined, providing information about which antigens act independently. Moreover, multivariate modeling may indicate patterns of response with stronger sensitivity and specificity than individual antigen responses. Most currently available methods for producing protein microarrays still require purification of proteins for printing on the array, or post-array protein identification. Our laboratory has developed a novel and unique method for producing protein microarrays that obviates these challenges by substituting the printing of cDNAs on the arrays, which are then transcribed and translated in situ as needed at the time of the assay. We have already used these microarrays to identify autoantibodies to tumor antigens in cancer patient sera, with equivalent sensitivity and specificity to standard ELISA. Here, we propose adapting our NAPPA protein microarray technology for use in the rapid and efficient screening of sera from cancer patients for antibodies to 1000 known and potential tumor antigens in a multiplex format for the early detection of breast cancer. Using tests sets and validation sets of serum samples derived from patients with early-stage breast cancer, we will determine the frequency, sensitivity and specificity of autoantibodies to tumor antigens for the early diagnosis of breast cancer. [unreadable] [unreadable]